The present invention relates to biocompatible films and gels formed from chemically modified polyanionic polysaccharides. In particular, the present invention relates to biocompatible, polymeric gels which are prepared by synthesizing the polymer in a mixed solvent system containing water and a water miscible solvent such as a lower alkanol, an alkyl pyrrolidones, DMSO or acetone.
Polyanionic polysaccharides are polysaccharides containing more than one negatively charged group, e.g., carboxyl groups at pH values above about 4.0. One such polyanionic polysaccharide, hyaluronic acid (xe2x80x9cHAxe2x80x9d), is a naturally occurring mucopolysaccharide found, for example, in synovial fluid, in vitreous humor, in blood vessel walls and the umbilical cord, and in other connective tissues The polysaccharide consists of alternating N-acetyl-D-glucosamine and D-glucuronic acid residues joined by alternating xcex2 1-3 glucuronidic and xcex2 1-4 glucosaminidic bonds, so that the repeating unit is -(1xe2x86x924)- xcex2-D-GlcA-(1xe2x86x923)-xcex2-D- GlcNAc-. In water, hyaluronic acid dissolves to form a highly viscous fluid. The molecular weight of hyaluronic acid isolated from natural sources generally falls within the range of 5xc3x97104 up to 1xc3x97107 daltons.
As used herein the term xe2x80x9cHAxe2x80x9d means hyaluronic acid, and any of its hyaluronate salts, including, for example, sodium hyaluronate (the sodium salt), potassium hyaluronate, agnesium hyaluronate, and calcium hyaluronate.
HA, in chemically modified (xe2x80x9cderivatizedxe2x80x9d) form, is useful as a surgical aid, to prevent adhesions or accretions of body tissues during the post-operation period. The derivatized HA gel or film is injected or inserted into the locus between the tissues that are to be kept separate to inhibit their mutual adhesion. To be effective the gel must remain in place and prevent tissue contact for a long enough time so that when the gel finally disperses and the tissues do come into contact, they will no longer have a tendency to adhere.
Chemically modified HA can also be useful for controlled release drug delivery. Balazs et al., 1986, U.S. Pat. No. 4,582,865, states that xe2x80x9ccross-linked gels of HA can slow down the release of a low molecular weight substance dispersed therein but not covalently attached to the gel macromolecular matrix.xe2x80x9d R. V. Sparer et al., 1983, Chapter 6, pages 107-119, in T. J. Roseman et al., Controlled Release Delivery Systems, Marcel Dekker, Inc., New York, describes sustained release of chloramphenicol covalently attached to hyaluronic acid via ester linkage, either directly or in an ester complex including an alanine bridge as an intermediate linking group.
I. Danishefsky et al., 1971, Carbohydrate Res., Vol. 16, pages 199-205, describes modifying a mucopolysaccharide by converting the carboxyl groups of the mucopolysaccharide into substituted amides by reacting the mucopolysaccharide with an amino acid ester in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (xe2x80x9cEDCxe2x80x9d) in aqueous solution. They reacted glycine methyl ester with a variety of polysaccharides, including HA. The resulting products are water soluble; that is, they rapidly disperse in water or in an aqueous environment such as is encountered between body tissues.
Proposals for rendering HA compositions less water soluble include cross-linking the HA. R. V. Sparer et al., 1983, Chapter 6, pages 107-119, in T. J. Roseman et al., Controlled Release Delivery Systems, Marcel Dekker, Inc., New York, describe modifying HA by attaching cysteine residues to the HA via amide bonds and then cross-linking the cysteine-modified HA by forming disulfide bonds between the attached cysteine residues. The cysteine-modified HA was itself water soluble and became water insoluble only upon cross-linking by oxidation to the disulfide form.
De Belder et al., PCT Publication No. WO 86/00912, describe a slowly-degradable gel, for preventing tissue adhesions following surgery, prepared by cross-linking a carboxyl-containing polysaccharide with a bi- or polyfunctional epoxide. Other reactive bi- or polyfunctional reagents that have been proposed for preparing cross-linked gels of HA having reduced water solubility include: 1,2,3,4-diepoxybutane in alkaline medium at 50.degree. C. (T. C. Laurent e al., 1964, Acta Chem. Scand., vol. 18, page 274); divinyl sulfone in alkaline 5 medium (E. A. Balasz et al., U.S. Pat. No. 4,582,865, (1986); and a variety of other reagents including formaldehyde, dimethylolurea, dimethylolethylene urea, ethylene oxide, a polyaziridine, and a polyisocyanate (E. A. Balasz et al., U.K. Patent Appl. No. 84 20 560 (1984). T. Malson et al., 1986, PCT Publication No. WO 86/00079, describe preparing cross-linked gels of HA for use as a vitreous humor substitute by reacting HA with a bi- or polyfunctional cross-linking reagent such as a di- or polyfunctional epoxide. T. Malson et al., 1986, EPO 0 193 510, describe preparing a shaped article by vacuum-drying or compressing a cross-linked HA gel.
The invention features an improved method for preparing a water insoluble gel by combining a polyanionic polysaccharide and an activating agent under conditions sufficient to form the gel. The reaction conditions of this invention include the use of an organic solvent which is selected from the group consisting of lower alkanols, alkyl pyrrolidones, DMSO and acetone. The organic solvents of this invention are miscible in water, and are present in the reaction medium in an amount of from about 5.0 % to about 80% by weight. A particularly preferred organic solvent is N-methylpyrrolidone, which is generally compatible with cardodiimide activating agents. N-methylpyrrolidone has a favorable biocompatibility profile in comparison to other organic solvents (rat LD50=4 g/Kg), and it can be used to obtain a high concentration of modified polyanionic polysaccharides having a low viscosity.
The use of a water miscible organic solvent in the reaction medium as described herein permits the polyanioinic polysaccharide to be synthesized using substantially less activating agent and at a significantly increased concentration of reactants, as compared with a similar reaction conducted in water alone. The increase in concentration of reactants can amount to as much as six fold or more, and the reduction in activating agent, e.g. carbodiimide, can be on the order of one-third or more. As an example, the use of N-methylpyrrolidone permits the concentration of the reaction solution to be as high as 4.0% (40 g/L), with yields of up to 82%, as compared to concentrations of 0.6% (6 g/L) and 64% yields without the use of N-methylpyrrolidone. Since carbodiimides are relatively expensive reagents, a reduction in carbodiimide usage of this level can represent a significant cost savings. Typically, the derivatization reaction requires the use of approximately 6 molar equivalence of EDC per mole of carboxyl group, and includes a precipitation step involving large quantities of ethanol. The use of ethanol precipitation in a subsequent purification procedure, which involves the use of large quantities of ethanol, can be eliminated by following the procedure of this invention.
Preferred polyanionic polysaccharides for use in the present invention include hyaluronic acid, carboxymethyl cellulose (xe2x80x9cCMCxe2x80x9d), carboxymethyl amylose (xe2x80x9cCMAxe2x80x9d), carboxymethyl chitosan, chondroitin-6-sulfate, dermatin sulfate, heparin, and heparin sulfate; CMC and CMA are particularly preferred.
The preferred activating agent is a carbodiimide, e.g., 1-ethyl-3 -(3-dimethylaminopropyl)carbodiimide or 1-ethyl-3-(3 -dimethylaminopropyl)carbodiimide methiodide.
The activating agent can be added to the polyanionic polysaccharide, or the polyanionic polysaccharide may be combined with the activating agent. Combinations of different polyanionic polysaccharides can also be used.
The preferred pH for carrying out the reaction is 4.0 to 5.0. The preferred concentration for the polysaccharide is 0.2M-2.0M. The molar ratio of carboxyl groups of polysaccharide to activating agent is preferably less than about 1:1, and more preferably less than about 1:6.
The gel may be provided in the form of an adhesion prevention composition, e.g., in the form of a composition suitable for incorporation in a syringe or laproscopic instrument for use in minimal invasive surgical procedures. The gel may also include a pharmaceutically active substance dispersed throughout it; in such cases, the gel is useful as a drug delivery system. Suitable substances include growth factors, enzymes, drugs, biopolymers, and biologically compatible synthetic polymers. Alternatively, the gel can be used in applications where viscoelastic supplementation is desired, such as in the phacoemulsion surgery for the removal of cataracts in eye surgery to minimize the damage to endothelial cells.
A xe2x80x9cbiocompatiblexe2x80x9d substance, as that term is used herein, is one that has no medically unacceptable toxic or injurious effects on biological function. A polyanionic polysaccharide which is reacted with a suitable activating agent forms a gel having decreased water solubility without the use of and separately added bi- or polyfunctional cross-linking reagents.
A xe2x80x9cwater insolublexe2x80x9d gel of the invention, as that phrase and like terms are used herein, is one formed using a 1% aqueous solution of polyanionic polysaccharide, modified according to the invention, having the same dimensions and similarly allowed to stand without stirring in a beaker of 50 ml of distilled water at 20xc2x0 C., remains structurally intact after 20 minutes, with the gel boundaries and edges still being present after 24 hours, although the gel is swollen.
A polyanionic polysaccharide is said to be xe2x80x9cactivatedxe2x80x9d, as that term is used herein, when it is treated in an aqueous mixture in a manner that renders the carboxyl groups on the polyanionic polysaccharide vulnerable to nucleophilic attack; and an xe2x80x9cactivating agentxe2x80x9d is a substance that, in an aqueous mixture including a polyanionic polysaccharide, causes the polyanionic polysaccharide to become so activated.
Because the gels are water insoluble, they can be thoroughly washed with water before use to remove unreacted substances. In addition, the gels can also be terminally stabilized by heat treatment without causing significant changes in the rheological properties of the gel prior to use.
The gels of the invention can also be prepared in colored form, by including a dye or stain in the reaction mixture. Such colored gels can be more easily seen when in place or during placement, making them easier to handle during surgical procedures than colorless ones.
The gels of the invention retain their strength even when hydrated. Because the gel adheres to biological tissues without the need for sutures, it is useful as postoperative adhesion prevention material. The gel can be applied to tissue even in the presence of bleeding.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.